Iron acquisition is essential for pathogenic bacteria. In the vertebrate host, however, iron is not readily available, and this scarcity restricts the growth of pathogenic microorganisms and limits their ability to infect and invade. Bacteria have evolved multiple high affinity iron acquisition systems to cope with iron-limiting environments. In spite of considerable characterization of these systems in the laboratory, for most pathogens, the sources of iron used in the host and the relative importance of the different acquisition systems within different niches of the host are poorly understood. The objectives of this study are to determine mechanisms by which gram-negative enteric pathogens obtain iron in the host and to study the possible transmission of iron-acquisition genes and their role in evolution of emerging pathogens. This study will focus on heme transport systems in Shigella and selected Escherichia coli, pathogens which are responsible for considerable morbidity and mortality throughout the world. The first Specific Aim is to continue the characterization of the heme transport locus from Shigella dysenteriae type 1. The PI has found that a nearly identical locus is present in many pathogenic E. coli, including E. coli 0157:H7, and the phylogenetic distribution of this locus is suggestive of horizontal gene transfer. Therefore, the second Specific Aim is to determine whether these genes are mobile and how they may be spreading within the enteric pathogens. The data and strains developed in characterizing the heme transport systems of Shigella and E. coli, together with previous data on high affinity iron transport systems, have placed the PI in the position to now accomplish the third Specific Aim, to determine how Shigella and E. coli acquire iron in vivo. The fourth Specific Aim is to use genetic approaches to understand which genes Shigella specifically expresses in vivo, and to elucidate the role of these genes in Shigella infection. Results obtained from this study will provide basic information on genetics and regulation of potential virulence factors in an important group of pathogens and will provide data useful in design of potential vaccine strains.